Excess NO Research Articles

Response of Asymmetric Dimethylarginine to Hemodialysis-Associated Hypotension in End-Stage Renal Disease Patients

Aims: To define the role of asymmetric dimethylarginine (ADMA) in the control of blood pressure (BP) during hemodialysis (HD). Methods:L-Arginine, ADMA and symmetric dimethylarginine (SDMA) levels of patients with (n = 18) or without (n = 13) hypotensive episodes during HD sessions were measured before and after HD treatment by liquid chromatography-mass spectrometry. Clinical variables, laboratory parameters and underlying pathologies of end-stage renal disease (ESRD) were comparable in the groups. BP was serially recorded. Results: In patients with ESRD, plasma dimethylarginines were markedly elevated and decreased significantly by the end of the HD sessions. ADMA levels in patients having hypotensive episodes during HD were significantly higher than in those maintaining their BP (before HD: 0.62 ± 0.11 µmol/l vs. 0.71 ± 0.13 µmol/l, p = 0.04; after HD: 0.31 ± 0.11 µmol/l vs. 0.43 ± 0.11 µmol/l, p = 0.01). There was a significant inverse relationship of the minimum systolic and diastolic BP during HD to the predialysis ADMA levels (for systolic BP r = –0.50, p < 0.01; for diastolic BP r = –0.59, p < 0.01) and to the postdialysis ADMA levels (for systolic BP r = –0.49, p < 0.01; for diastolic BP r = –0.51, p < 0.005), respectively. Conclusions: It is suggested that excessive NO generation is involved in the HD-associated hypotension and induces an increase in plasma ADMA levels to prevent further fall in BP.

Study of a Fe–zeolite-based system as NH 3-SCR catalyst for diesel exhaust aftertreatment

The reactivity of a commercial Fe-exchanged zeolite catalyst is herein analyzed in the NH 3-SCR (selective catalytic reduction) reactions of interest for industrial applications to the aftertreatment of diesel exhausts. Transient tests, addressing reacting systems of growing complexity (NH 3 + O 2, NO + O 2, NO 2 + O 2, NH 3 + NO + O 2, NH 3 + NO 2 + O 2 and NH 3 + NO + NO 2 + O 2) in a representative temperature range, were performed over the catalyst both in the powder form, in order to derive intrinsic kinetic information, and in the form of small core washcoated monoliths. Results were compared to those collected over a commercial extruded V 2O 5–WO 3/TiO 2 catalyst. The zeolite system was characterized by: (i) a higher NH 3 storage capacity, (ii) a higher activity in NH 3 and NO oxidation reactions, (iii) a higher activity in the standard SCR reaction at low temperature, (iv) a higher activity in the fast SCR reaction and (v) a higher selectivity to N 2O in the presence of excess NO 2.

Role of extracellular signal-regulated kinases (ERK) in leptin-induced hypertension

We investigated if extracellular signal-regulated kinases (ERK) and oxidative stress are involved in the pathogenesis of arterial hypertension induced by chronic leptin administration in the rat. Leptin was administered at a dose of 0.25 mg/kg twice daily s.c. for 4 or 8 days. Blood pressure (BP) was higher in leptin-treated than in control animals from the third day of the experiment. The superoxide dismutase (SOD) mimetic, tempol, normalized BP in leptin-treated rats on days 6, 7 and 8, whereas the ERK inhibitor, PD98059, exerted a hypotensive effect on days 3 through 6. Leptin increased ERK phosphorylation level in renal and aortic tissues more markedly after 4 than after 8 days of treatment. In addition, leptin reduced urinary Na+ excretion and increased renal Na+,K+-ATPase activity, and these effects were abolished on days 4 and 8 by PD98059 and tempol, respectively. The levels of NO metabolites and cGMP were reduced in animals receiving leptin for 8 days. Markers of oxidative stress (H2O2 and lipid peroxidation products) were elevated to a greater extent after 4 than after 8 days of leptin treatment. In contrast, nitrotyrosine, a marker of protein nitration by peroxynitrite, was higher in animals receiving leptin for 8 days. NADPH oxidase inhibitor, apocynin, prevented leptin's effect on BP, ERK, Na+,K+-ATPase/Na+ excretion and NO formation at all time points. SOD activity was reduced, whereas glutathione peroxidase (GPx) activity was increased in the group treated with leptin for 8 days. These data indicate that: (1) ERK, activated by oxidative stress, is involved only in the early phase of leptin-induced BP elevation, (2) the later phase of leptin-induced hypertension is characterized by excessive NO inactivation by superoxide, (3) the time-dependent shift from ERK to O2−-NO dependent mechanism may be associated with reduced SOD/GPx ratio, which favors formation of O2− instead of H2O2.

Protective Effect of Polysaccharide Fractions from Radix A. Sinensis against tert-Butylhydroperoxide Induced Oxidative Injury in Murine Peritoneal Macrophages

Three Angelica sinensis polysaccharide fractions (APFs), named APF1, APF2 and APF3, were isolated and purified from Radix A. sinensis and their antioxidant activities were evaluated in isolated mouse peritoneal macrophages by pretreatment with APFs before exposure to 0.2 mM tertbutylhydroperoxide (t-BHP). The results showed that pretreatment of the macrophages with APFs as low as 10 microg/ml could significantly enhance t-BHP-decreased cell survival, intracellular glutathione (GSH) content and superoxide dismutase (SOD) activity, and also inhibited t-BHP-increased lactate dehydrogenase (LDH) leakage and malondialdehyde (MDA) formation (p < 0.05), and APF3 was the most active fraction, followed by APF2 and APF1 in decreasing order. Furthermore, we found for the first time that the bound-protein in APF3 was associated closely with the protective effects and the polysaccharide inhibited the excess NO release from t-BHP-activated macrophages to protect host cells.

Activation of signaling molecules and matrix metalloproteinases in right ventricular myocardium of rats with pulmonary hypertension

Pulmonary hypertension induces right ventricular (RV) overload, which is transmitted to cardiomyocytes via integrins that activate intracellular messengers, including focal adhesion kinase (FAK) and neuronal nitric oxide synthase (NOS1). We investigated whether RV hypertrophy (RVH) and RV failure (RVF) were associated with activation of FAK, NOS1, and matrix metalloproteinases (MMPs). Rats were treated without (RVC) or with a low dose of monocrotaline (30 mg/kg) to induce RVH, and with a high dose (80 mg/kg) to induce RVF. After ≈30 days, RV function was determined using a combined pressure-conductance catheter. After sacrifice, FAK, NOS1, their phosphorylated forms (FAK-P and NOS1-P), MMP-2, and MMP-9 were quantified in RV myocardium by immunohistochemistry. In RVH and RVF, RV weight/ body weight increased by 36% and 109%, whereas RV ejection fraction decreased by 23% and 57% compared to RVC, respectively. FAK-P and FAK-P/FAK were highest in RVH (2.87±0.12 and 2.52±0.23 fold compared to RVC, respectively) and slightly elevated in RVF (1.76±0.17 and 1.15±0.13 fold compared to RVC, respectively). NOS1-P and NOS1-P/NOS1 were increased in RVH (1.63±0.12 and 3.06±0.80 fold compared to RVC, respectively) and RVF (2.16±0.03 and 3.30±0.38 fold compared to RVC, respectively). MMP-2 was highest in RVH and intermediate in RVF (3.50±0.12 and 1.84±0.22 fold compared to RVC, respectively). MMP-9 was elevated in RVH and RVF (2.39±0.35 and 2.92±0.68 fold compared to RVC, respectively). Activation of FAK in RVH points to an integrin-dependent hypertrophic response of the myocardium. Activation of NOS1 in failing RV suggests a role of excessive NO in the development of failure and activation of MMPs leading to ventricular remodeling.

Abstract 1228: Decreased Mitochondrial Biogenesis and Increased Pro-oxidant Activity Are Associated with Oxidative Impairment of Complex II in the Postischemic Heart

Increased O 2 ·− generated in mitochondria and NO is a key mechanism of cell death in the postischemic injury. Succinate ubiquinone reductase (SQR or Complex II) is a crucial segment of electron transport chain. Oxidative impairment of SQR has been observed in the postischemic myocardium. When rats are subjected to a 30 min coronary ligation, followed by a 24 h reperfusion, two polypeptides in tissue homogenate were found to react with the antibody against nuclear-encoded 29 kDa iron-sulfur protein (ISP) of SQR. One at 29 kDa corresponded to the mature form localized in mitochondria was present in normal amounts. The other with higher molecular weight 32 kDa corresponded to the precursor localized in cytosol was marked deficient. Similar results were observed when isolated heart was subjected to global ischemia (30min) and reperfusion (1h). Taken together, these results implicate mitochondria myopathy with an abnormality of biogenesis in the postischemic heart. The SQR mRNA level analyzed by quantitative real-time PCR was decreased 51%. Both mRNA and protein expressions of transcription coactivator PGC-1α were decreased in postischemic models 44% and 38% respectively, indicating a defect in the regulation and coordination of mitochondrial biogenesis. The mRNA levels of downstream transcription factors and antioxidant enzymes including NRF, TFAM, MnSOD, and UCP were similarly decreased; indicating that defect in mitochondrial biogenesis augments the oxidative stress in the postischemic heart. In addition, peroxynitrite formation was detected in the mitochondria of postischemic myocyte. The SQR isolated from myocardium was further used to study the interaction of NO with SQR in vitro . Under the conditions of enzyme turnover, SQR obtained after NO (25 fold excess) pretreatment gained catalytic function to generate hydroxyl radical detected by EPR spin trapping using DEPMPO. In the presence of catalase and iron chelator, the EPR signal of associated with DEPMPO/ · OH was completely abolished, suggesting the involvement of iron-H 2 O 2 dependent Fenton reaction. Direct EPR measurement at 77 °K indicated the formation of nonheme iron-NO complex, implying electron leakage to O 2 had occurred at the ISP of SQR and excess NO predisposed SQR to pro-oxidant activity.

THE GREEN TEA POLYPHENOL EPIGALLOCATECHIN-3-GALLATE IMPROVES SYSTEMIC HEMODYNAMICS AND SURVIVAL IN RODENT MODELS OF POLYMICROBIAL SEPSIS

Epigallocatechin-3-gallate (EGCG) is the main polyphenolic flavonoid found in green tea. Recent in vitro studies have suggested that EGCG inhibits activation of the nuclear factor-kappaB (NF-kappaB) pathway. The NF-kappaB is a transcriptional factor required for gene expression of many inflammatory mediators, including the inducible isoform of nitric oxide synthase (NOS2). Excessive NO production by NOS2 is directly linked to the vasoplegia, shock, and mortality associated with sepsis. Accordingly, we hypothesized that EGCG administration would inhibit NOS2 gene expression and thereby improve survival in a rodent model of polymicrobial sepsis. Polymicrobial sepsis was induced in male Sprague-Dawley rats (hemodynamic study) and C57BL6 mice (mortality study) via cecal ligation and double puncture (CL2P). Rodents were treated with either EGCG (10 mg/kg intraperitoneally) or vehicle at 1 and 6 h after CL2P and every 12 h thereafter. In the hemodynamic study, mean arterial blood pressure was monitored for 18 h, and rats were killed at 3, 6, and 18 h after CL2P. In the mortality study, survival was monitored for 72 h after CL2P in mice. In vehicle-treated rodents, CL2P was associated with profound hypotension and greater than 80% mortality rate. Epigallocatechin-3-gallate treatment significantly improved both the hypotension and survival. In vitro experiments further showed that EGCG inhibited activation of NF-kappaB and subsequent NOS2 gene expression in a primary culture of rat aortic smooth muscle cells. Epigallocatechin-3-gallate may therefore represent a potential nutritional supplement or pharmacologic agent in patients with sepsis.

Expression of neuronal nitric oxide synthase in the retina of a rat model of chronic glaucoma

We investigated the expression of neuronal nitric oxide synthase (nNOS) in a rat retina model of chronic glaucoma, which was produced by electrocauterization of the episcleral vessels. Western-blot analysis showed that nNOS expression was significantly increased in cauterized retinas. nNOS immunoreactivity was observed in the cells of both the inner nuclear layer and the ganglion cell layer. Double labeling of retinal ganglion cells (RGCs) revealed that RGCs in the retina of cauterized rat was nNOS-immunopositive. Systemic administration of l-NAME ( N G-nitro- l-arginine-methyl-ester), a non-specific NOS inhibitor, reduced RGC loss in cauterized rat retina, but there was no statistical significance ( P = .06). These results suggest that the cytotoxicity of excessive NO plays a role in selective RGC loss in glaucoma.

Resolution of the Spectroscopy versus Crystallography Issue for NO Intermediates of Nitrite Reductase from Rhodobacter sphaeroides

Copper nitrite reductase contains a T1 and T2 Cu center and catalyzes the one electron reduction of nitrite to NO. This study determines that reaction of the reduced enzyme with excess NO generates the T1 reduced T2 oxidized-nitrite bound form and not the η2-NO·Cu+ species observed in the crystal structure. Reaction intermediates have been trapped and N2O gas detected during this reaction, allowing elucidation of the mechanism by which both the T2 and NO are oxidized by the reaction of NO with the fully reduced enzyme. Density functional theory calculations provide insight into factors which favor the η2-NO·Cu+ complex in the crystal structure.

Gemfibrozil Ameliorates Relapsing-Remitting Experimental Autoimmune Encephalomyelitis Independent of Peroxisome Proliferator-Activated Receptor-α

The present study underlines the importance of gemfibrozil, a lipid-lowering drug and an activator of peroxisome proliferator-activated receptor-alpha (PPAR-alpha), in inhibiting the disease process of adoptively transferred experimental allergic encephalomyelitis (EAE), an animal model of relapsing-remitting multiple sclerosis. Clinical symptoms of EAE, infiltration of mononuclear cells, and demyelination were significantly lower in SJL/J female mice receiving gemfibrozil through food chow than those without gemfibrozil. It is noteworthy that the drug was equally effective in treating EAE in PPAR-alpha wild-type as well as knockout mice. Gemfibrozil also inhibited the encephalitogenicity of MBP-primed T cells and switched the immune response from a Th1 to a Th2 profile independent of PPAR-alpha. Gemfibrozil consistently inhibited the expression and DNA-binding activity of T-bet, a key regulator of interferon-gamma (IFN-gamma) expression and stimulated the expression and DNA-binding activity of GATA3, a key regulator of IL-4. Gemfibrozil treatment decreased the number of T-bet-positive T cells and increased the number of GATA3-positive T cells in spleen of donor mice. The histological and immunohistochemical analyses also demonstrate the inhibitory effect of gemfibrozil on the invasion of T-bet-positive T cells into the spinal cord of EAE mice. Furthermore, we demonstrate that the differential effect of gemfibrozil on the expression of T-bet and GATA3 was due to its inhibitory effect on NO production. Although excess NO favored the expression of T-bet, scavenging of NO stimulated the expression of GATA-3. Taken together, our results suggest gemfibrozil, an approved drug for hyperlipidemia in humans, may find further therapeutic use in multiple sclerosis.

Dynamic ligand exchange as mechanism for sGC selectivity, NO activation and its reversal

Nitric oxide is the main physiologic ligand of soluble gua-nylyl cyclase (sGC), a hemoprotein with high affinity forNO. Purified sGC binds NO with an estimated picomolarKd for NO, which corresponds to only a few molecules ofNO per cell. The His105 residue of the β subunit is theproximal ligand of the heme (His105-heme complex witha 430 nm Soret band). Binding of NO to the sGC hemeforms a six coordinate (6C) complex (His105-heme-NO),which is then converted into a five coordinate (5C) com-plex (His105 and heme-NO) [1]. This transformationcoincides with several hundred fold activation of thecyclase activity. The enzyme displays NO-concentrationdependence not only in the NO binding, but also in the6C sGC-NO →5C sGC-NO complex conversion [1]. Previ-ous studies showed that the sGC-NO complex obtainedunder stoichiometric NO amounts is not fully activated,while addition of GTP substrate or additional NO resultsin a fully activated sGC [2].In this current report we investigated the molecular basisof this NO- and GTP-dependence of enzyme activation.Stop-flow techniques were used to monitor the kinetics ofNO-sGC complex formation and its fate under stoichio-metric and excess NO conditions or in the presence ofGTP. Under both stoichiometric and excess NO condi-tions 6C His105-heme-NO complex (~420 nm Soretband) is formed at a diffusion limited rate independentlyof the presence or absence of GTP. Although in all testedconditions the His105-heme-NO complex is convertedinto five coordinate complex (free His105 and heme-NOcomplex; 399 nm Soret band), the fate of this complexdepends on the amount of NO applied or the presence ofGTP substrate.Under stoichiometric conditions, the heme-NO revertsback to a 430 nm species with sub-second kinetics, pre-sumably due to the loss of NO. The newly formed His105-heme complex (sGC*) may rebind NO and forms a 5CsGC-NO complex (399 nm species), but with muchslower kinetics (minutes vs. sub-millisecond kinetics forintial sGC). This secondary sGC-NO* complex is, mostprobably, the weakly activated sGC-NO complexdescribed previously [2]. However, even a two-fold molarexcess NO or the presence of GTP generates a 5C NO-heme complex with no sign of NO loss.Spectral properties of the sGC* state generated after theloss of NO (430 nm Soret peak) indicate that the hememoiety remains coordinated by a histidine residue. Differ-ent NO-binding properties, however, suggest that signifi-cant changes in the properties of the enzyme occurred. Weinvestigated whether a change of proximal ligands mayexplain such transformation during the conversion of 5CsGC-NO to sGC*. We substituted the βHis107 position,which is conserved in all mammalian β1 subunits, to leu-cine. Similar to wild type sGC mutant αβHis107→Leuenzyme also contains heme with a Soret peak at 430 nm.The mutant has different NO-binding kinetics, since itforms a stable sGC-NO complex, which does not looseNO even under sub-stoichiometric NO conditions.

Effect of Nitric Oxide on Ram Sperm Motility In vitro

The aim of this study is to investigate the effects of NO on sperm motility of ram. After incubation of normozoospermic samples for 160 min in the presence of L-arginine (NOS substrate), SNP (NO donor), L-NAME (non-selective nitric oxide inhibitor) and packed erythrocytes (NO scavenger), sperm motility assessed in four grades (A, B, C, D). In this study, L-arginine and SNP non-significantly increased progressive motility at low concentration and significantly decreased progressive motility at high concentration. L-NAME and packed erythrocytes dose-dependently decreased progressive sperm motility. It is concluded that sperm motility of ram physiologically depend on nitric oxide action although excess generation of nitric oxide by sperm or exogenous NO could not increase sperm motility and it seems that excess NO provides toxic condition to decrease sperm motility.

A CFD assisted control system design with applications to NO x control in a FGR furnace

In this paper, a novel technique to design control systems for industrial processes with non-linear distributed parameters is proposed. The technique utilizes computational fluid dynamics (CFD) simulation to extract the most essential characteristics from the non-linear industrial process, and then represent them as a set of linear dynamic models around a specific operating point. Based on the linear dynamic representation, a closed-loop feedback linear control system can be designed to maintain the desired performance for the system around the chosen operating point. To illustrate such a design process, an industrial reheating furnace with flue gas recirculation (FGR) is selected herein. The method involves the numerical solution of the partial differential equations describing the fluid flow, heat transfer and combustion process in the furnace. The resulting dynamic relations between the furnace inputs and outputs can then be represented in terms of a multi-input and multi-output transfer function matrix. The objective of the control system is then to maintain the optimally selected furnace operating conditions and compensate for any deviations caused by disturbances to minimize the nitric oxides (NO x ) emission through feedback mechanisms. The performance of the closed-loop controlled furnace is evaluated not only in the linear domain, but also with the detailed full-scale non-linear CFD model. The results have shown that the proposed method is viable and the designed control system can indeed minimize the deviation of the furnace from the desired operating conditions and hence to prevent any excessive NO x formation in the combustion process.

Correlation between Flavonoid Content and the NO Production Inhibitory Activity of Peel Extracts from Various Citrus Fruits

We investigated the correlation between the flavonoid content and NO production inhibitory activity of fruit peel extracts using 20 citrus plants. The contents of seven flavonoids (naringin, naringenin, hesperidin, hesperetin, rutin, nobiletin, and tangeretin) were determined by HPLC analysis. Each citrus peel extract varied in flavonoid content, but the contents of nobiletin and tangeretin, which were contained in all 20 fruit peels, showed a positive and significant correlation with each other (r=0.879, p<0.0005 for immature fruit peels; r=0.858, p<0.0005 for mature fruit peels). All citrus peel extracts dose-dependently inhibited LPS-induced NO production in RAW 264.7 cells. This inhibitory effect was significantly and positively correlated with the content of nobiletin and tangeretin. Nobiletin showed a more potent NO production inhibitory activity (IC50=26.5 microM) compared to tangeretin (IC50=136.6 microM). This result supports the premise that nobiletin-rich citrus may provide protection against disease resulting from excessive NO production.

Dissociation of Nitric Oxide from Soluble Guanylate Cyclase and Heme-Nitric Oxide/Oxygen Binding Domain Constructs

Regulation of soluble guanylate cyclase (sGC), the primary NO receptor, is linked to NO binding to the prosthetic heme group. Recent studies have demonstrated that the degree and duration of sGC activation depend on the presence and ratio of purine nucleotides and on the presence of excess NO. We measured NO dissociation from full-length alpha1beta1 sGC, and the constructs beta1(1-194), beta1(1-385), and beta2(1-217), at 37 and 10 degrees C with and without the substrate analogue guanosine-5'-[(alpha,beta-methylene]triphosphate (GMPCPP) or the activator 3-(5'-hydroxymethyl-3'-furyl)-1-benzylindazole (YC-1). NO dissociation from each construct was complex, requiring two exponentials to fit the data. Decreasing the temperature decreased the contribution of the faster exponential for all constructs. Inclusion of YC-1 moderately accelerated NO dissociation from sGC and beta2(1-217) at 37 degrees C and dramatically accelerated NO dissociation from sGC at 10 degrees C. The presence of GMPCPP also dramatically accelerated NO dissociation from sGC at 10 degrees C. This acceleration is due to increases in the observed rate for each exponential and in the contribution of the faster exponential. Increases in the contribution of the faster exponential correlated with higher activation of sGC by NO. These data indicate that the sGC ferrous-nitrosyl complex adopts two 5-coordinate conformations, a lower activity "closed" form, which releases NO slowly, and a higher activity "open" form, which releases NO rapidly. The ratio of these two species affects the overall rate of NO dissociation. These results have implications for the function of sGC in vivo, where there is evidence for two NO-regulated activity states.

Component and Antioxidant Properties of Polysaccharide Fractions Isolated from Angelica sinensis (OLIV.) DIELS

An analytical method of high performance capillary electrophoresis (HPCE) was developed to simultaneously separate and identify the component monosaccharides of Angelica sinensis polysaccharide fractions (APFs), named APF1, APF2 and APF3. The predominant sugars in APFs were identified as arabinose, glucose, rhamnose, galactose and galacturonic acid as well as trace amount of mannose and glucuronic acid, and the fractionation altered significantly the distribution of component monosaccharides in APFs. APF3 was the most active fraction to effectively inhibit H(2)O(2)-caused decrease of cell viability, lactate dehydrogenase (LDH) leakage and malondialdehyde (MDA) formation, and also reduced H(2)O(2)-caused decline of superoxide dismutase (SOD) activity and glutathione (GSH) depletion (p<0.05), followed by APF2 and APF1 in decreasing order. Furthermore, it was found that APFs (100 microg/ml) could protect macrophages by inhibiting the release of excess NO and reactive oxygen species (ROS) induced by high concentrations of H(2)O(2) (0.8-1.6 mM).

Reductive nitrosylation and peroxynitrite‐mediated oxidation of heme–hemopexin

Hemopexin (HPX), which serves as a scavenger and transporter of toxic plasma heme, has been postulated to play a key role in the homeostasis of NO. In fact, HPX-heme(II) reversibly binds NO and facilitates NO scavenging by O(2). HPX-heme is formed by two four-bladed beta-propeller domains. The heme is bound between the two beta-propeller domains, residues His213 and His266 coordinate the heme iron atom. HPX-heme displays structural features of heme-proteins endowed with (pseudo-)enzymatic activities. In this study, the kinetics of rabbit HPX-heme(III) reductive nitrosylation and peroxynitrite-mediated oxidation of HPX-heme(II)-NO are reported. In the presence of excess NO, HPX-heme(III) is converted to HPX-heme(II)-NO by reductive nitrosylation. The second-order rate constant for HPX-heme(III) reductive nitrosylation is (1.3 +/- 0.1) x 10(1) m(-1).s(-1), at pH 7.0 and 10.0 degrees C. NO binding to HPX-heme(III) is rate limiting. In the absence and presence of CO2 (1.2 x 10(-3) m), excess peroxynitrite reacts with HPX-heme(II)-NO (2.6 x 10(-6) m) leading to HPX-heme(III) and NO, via the transient HPX-heme(III)-NO species. Values of the second-order rate constant for HPX-heme(III)-NO formation are (8.6 +/- 0.8) x 10(4) and (1.2 +/- 0.2) x 10(6) m(-1).s(-1) in the absence and presence of CO2, respectively, at pH 7.0 and 10.0 degrees C. The CO2-independent value of the first-order rate constant for HPX-heme(III)-NO denitrosylation is (4.3 +/- 0.4) x 10(-1) s(-1), at pH 7.0 and 10.0 degrees C. HPX-heme(III)-NO denitrosylation is rate limiting. HPX-heme(II)-NO appears to act as an efficient scavenger of peroxynitrite and of strong oxidants and nitrating species following the reaction of peroxynitrite with CO2 (e.g. ONOOC(O)O-, CO3-, and NO2).

Down-regulation of CXCR2 on Neutrophils in Severe Sepsis Is Mediated by Inducible Nitric Oxide Synthase–derived Nitric Oxide

The failure of neutrophils to migrate to an infection focus during severe sepsis is an important determinant of the inability of a host to deal with an infectious insult. Our laboratory has shown that inducible nitric oxide synthase (iNOS) induction and NO production contribute to the failure of neutrophils to migrate in the context of sepsis. We investigated whether CXCR2 expression contributed to the failure of neutrophils to migrate during severe sepsis and the role of NO in modulating CXCR2 expression on neutrophils in mice subjected to nonsevere (NS) or severe (S) cecal ligation and puncture (CLP). Neutrophil migration to the infection focus was deficient in S-CLP mice, a phenomenon prevented by pharmacologic (aminoguanidine, l-canavanine) or genetic (iNOS gene deletion) inhibition of iNOS. The expression of CXCR2 on neutrophils from S-CLP mice was significantly reduced when compared with neutrophils from NS-CLP or sham-operated mice. CXCR2 expression was reestablished by pharmacologic and genetic inhibition of iNOS. Immunofluorescence and confocal analysis revealed that iNOS blockade reduced neutrophil CXCR2 internalization. Adhesion and emigration of neutrophils in macrophage inflammatory protein-2-stimulated mesentery microcirculation were reduced in S-CLP mice, compared with NS-CLP mice, and reestablished by pretreatment with aminoguanidine or l-canavanine. The NO donor S-nitroso-N-acetyl-d,l-penicillamine inhibited CXCL8-induced human neutrophil chemotaxis and CXCR2 expression on human and murine neutrophils. These results highlight evidences that the failure of neutrophils to migrate to an infection focus during severe sepsis is associated with excessive NO production and NO-dependent regulation of the expression of CXCR2 on the neutrophil surface.

Protective effect of ischemic preconditioning on rat small-for-size liver graft and its mechanism

目的 探讨缺血预处理(IPC)对大鼠小体积供肝的保护作用及其机制.方法 120只SD大鼠随机分为3组(每组20对):无热缺血组(NWI)、缺血再灌注组(WI)和缺血预处理组(IPC).用双袖套法建立大鼠小体积肝移植模型.各组10只受体大鼠于术前1 d、术后1、2、3、5 d取血,用自动生化分析仪检测AST和ALT.NWI组于供肝灌注前及植入后0.5、1、2、3 h,WI组于热缺血前及植入后0.5、1、2、3 h,IPC组于IPC前、IPC后及植入后0.5、1、2、3 h取肝组织,用硝酸还原法检测其NO浓度.结果 IPC可降低大鼠小体积肝移植术后血清AST和ALT浓度,提高再灌注早期肝脏组织NO的浓度,降低再灌注晚期肝脏组织NO的浓度(P＜0.05).结论 NO在大鼠肝脏的缺血再灌注损伤中可能具有双重作用.IPC对大鼠小体积供肝的缺血再灌注损伤有保护作用.其机制可能是通过促进供肝再灌注后早期NO合成,改善肝脏微循环,同时抑制供肝再灌注后晚期NO合成,减轻过量NO的损伤作用,从而保护移植肝脏功能。

Luminescent Properties of Water-Soluble Conjugated Metallopolymers and Their Application to Fluorescent Nitric Oxide Detection

Water-soluble pi-conjugated polymers (CPs) incorporating 5,5'-(2,2'-dipyridyl) (CP1) or 6,6'-(2,2'-dipyridyl) (CP2) units within the pi-conjugated backbone were prepared as scaffolds for macromolecular metal complexation. The response of CP emission to a range of metal ions was investigated in water, 10 mM aqueous sodium dodecyl sulfate, and acetonitrile/water (95:5). Cupric ions are the most efficient quenchers of CP emission, with K(SV) = 1.1 x 10(5) and 5.2 x 10(4) M(-1) in water for CP1a (40% bipyridyl monomer units) and CP1b (20% bipyridyl monomer units), respectively. Quenching is approximately twice as effective in acetonitrile/water (95:5) (K(SV) = 3.1 x 10(5) M(-1) for CP1a and 1.1 x 10(5) M(-1) for CP1b). Partial restoration of emission was observed upon exposure of Cu(II)-CP solutions to excess NO(g) in acetonitrile/water (95:5) or 10 mM SDS(aq).